Copolymers containing lactic acid and glycolic acid residues (poly(lactic acid-co-glycolic acid) or PLGA) have been used for sutures, structural implants, and as drug delivery vehicles since the 1970s. The use of these biodegradable polymers for drug delivery is reviewed by Brannon-Peppas, L. and Vert, M. “Polylactic and Polyglycolic Acids as Drug Delivery Carriers” in Handbook of Pharmaceutical Controlled Release Technology, Wise, D. L. ed., Marcel Dekker, New York, pp. 99–130, 2000. The preparation of PLGA polymers for drug delivery applications by methods such as single emulsion, double emulsion, phase separation, and spray drying is reviewed by Jain et al., Drug Development and Industrial Pharmacy, Vol. 24, pp 703–727, 1998. Examples of early work on the use of PLGA prepare films and to prepare spray dried particles were described in U.S. Pat. Nos. 3,773,919, 3,887,699, and 4,293,339 and examples of early work on the preparation of polylactic acid or PLGA microcapsules for the release of biologically active compounds were described in T. Chang, J. Bioeng., Vol 1, pp 25–32, 1976 and in U.S. Pat. No. 4,675,189.
The incorporation of proteins into PLGA microparticles and the release profiles of these proteins from the microparticles into physiological solutions has been intensively investigated, as reviewed by Roskos, K. V. and Maskiewicz, R. “Degradable Controlled Release Systems Useful for Protein Delivery” in Protein Delivery: Physical Systems, Sanders and Hendren eds., Plenum press, New York, pp. 45–92, 1997 and Putney, S. D., Current Opinion in Chemical Biology, Vol 2, pp 548–552, 1998.
Major problems associated with the release of protein therapeutics from biodegradable PLGA particles include the stability of the protein, both during incorporation in the PLGA particle and during release (reviewed by van de Weert et al., Pharmaceutical Research, Vol 17, pp. 1159–1167, 2000 and Burke, P. “Controlled Release Protein Therapeutics: Effects of Process and Formulation on Stability” in Handbook of Pharmaceutical Controlled Release Technology, Wise, D. L. ed., Marcel Dekker, New York, pp. 661–692, 2000), and the ubiquitous presence of a “burst” effect, where a significant fraction of the protein incorporated in the microparticle is released immediately after the particle is introduced into a physiological solution, followed by a much slower rate of long term protein release (reviewed by Crotts, G. and Park, T. G., Journal of Microencapsulation, Vol. 15, pp. 699–713, 1998; Sandor et al., Journal of Controlled Release, Vol. 76, pp. 297–311, 2001; and Huang, X. and Brazel, C. S., Journal of Controlled Release, Vol. 73, pp. 121–136, 2001).
There are many reports in the literature of the use of hydrophilic gels to protect proteins from inactivation during PLGA microsphere formation and to the use of these gels to modify the protein release profile in a physiological solution. The following are examples of protein-loaded gels that researchers have incorporated into PLGA microparticles: gelatin nanoparticles (Li et al., Journal of Pharmaceutical Sciences, Vol. 86, pp. 891–895, 1997), agarose (Wang, N, and Wu, X. S., International Journal of Pharmaceutics, Vol. 166, pp. 1–14, 1998), dextran, heparin, alginate, or bovine serum albumin (Sanchez et al., International Journal of Pharmaceutics, Vol 185, pp. 255–266, 1999), poly(vinyl alcohol) (Wang et al., Pharmaceutical Research, Vol. 16, pp 1430–1435, 1999), Poly(acryloyl hydroxyethyl starch) (Woo et al., Pharmaceutical Research, Vol. 18, pp 1600–1606, 2001), and hydroxypropyl cellulose (Lee et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., Vol 23, pp. 333–334, 1996). These literature reports indicate that the use of hydrophilic gels can, in some cases, increase the incorporation of protein into the PLGA particle, increase the stability of the protein in the PLGA particle, and increase the total amount of protein released from the PLGA particle. In no case, however, did addition of the gel prevent the initial burst of protein release that occurred when the particles were suspended in a physiological solution. In many of these cases, the stability of the protein has not been determined.
Neutral surfactants such as Tween (Cleland, J. L. and Jones, A. J. S., Pharmaceutical Research, Vol. 13, pp. 1464–1475, 1996), poly(ethylene glycol) (Pean, J.-M. et al., Pharmaceutical Research, Vol. 16, pp. 1294–1299, 1999), γ-hydroxypropyl cyclodextrin (Johansen, P. et al., Pharmaceutical Research, Vol. 15, pp. 1103–1110, 1998), and polyoxamer (De Rosa et al., Journal of Controlled Release, Vol. 69, pp. 283–295, 2000) have been added to the aqueous protein phase used to make PLGA particles. Surfactants such as Pluronic™ (Park et al., Macromolecules, Vol. 25, pp. 116–122, 1992) and poly(ethylene glycol) (Jiang, W. and Schwendeman, S. P., Pharmaceutical Research, Vol. 18, pp. 878–885. 2001) have been added to the organic PLGA phase during particle preparation. In some cases, the addition of surfactants stabilizes the protein to emulsification processes and increases the total amount of protein released into physiological solution. In no case, however, do these surfactants remove the burst effect or change the release profile so that the amount of protein released into solution is constant over time.
The incorporation of charged surfactants into PLGA particles has been described in U.S. Pat. No. 5,985,309. In this case, the inventors incorporated charged therapeutic agents and surfactants with the opposite charge in their PLGA formulations. Quaternary ammonium surfactants were incorporated into PLGA particles and shown to increase the percentage release for small molecular weight therapeutic agents (U.S. Pat. No. 5,759,583).
Polysaccharide hydrogels have been extensively investigated as possible vehicles for protein delivery, as reviewed by Chen, J. et al., Carbohydrate Polymers, Volume 28, pp. 69–76, 1995. Particles of polysaccharides have been prepared with proteins or small molecular weight drugs and then coated with cationic molecules. For example, alginate particles have been coated with polylysine, poly(ethylene imine) or chitosan (Wheatley et al., Journal of Applied Polymer Science, Vol 43, pp. 2123–2135, 1991; Austin et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., Vol 23, pp. 739–740, 1996; Gombotz, W. R. and Wee, S. F., Advanced Drug Delivery Reviews, Vol, 31, pp. 267–285, 1998; Sezer, A. D. and Akbuga, J., J. Microencapsulation, Vol. 16, pp. 195–203, 1999; U.S. Pat. No. 6,238,705 B1) and carrageenan particles have been coated with oligoamines, diamines, and monamines (Patil, R. T. and Speaker, T. J., Journal of Pharmaceutical Sciences, Vol 89, pp. 9–15, 2000). In most cases proteins are released from these particles in hours or days, although longer release times have been proposed. Prokop et al., (Advances in Polymer Science, Vol, 136, pp. 53–73, 1998) report the coating of multiple polysaccharide polyanion blends with multiple polycation blends in order to microencapsulate cells for immuno-isolation. It has been reported that anionic polysaccharide films, foams, and fibers can be spray coated or brush coated with PLGA (U.S. Pat. No. 6,294,202 B1).
The use of gum arabic to stabilize lyophilized protein is described in the European Patent application #EP0950663A1 and in the U.S. Pat. No. 6,391,296 and the use of high gum arabic concentrations to stabilize proteins in solution is described in our U.S. patent application Ser. No. #12988. Complex coacervates have been made from gum arabic and cationic proteins, which have been spray dried to form microparticles (Burgess, D. J. and Ponsart, S., Journal of Microencapsulation, Vol. 15, pp. 569–579, 1998). The enzyme β-glucuronidase was encapsulated in this system in the absence of PLGA. Approximately 30% of the encapsulated enzyme was released in a burst when these particles were added to a physiological buffer and an additional 30% of the enzyme was released with a linear rate over 12 days.
U.S. Pat. No. 6,294,202 describes the compositions of anionic polysaccharides in bioabsorbable polymers. U.S. Pat. No. 5,700,486 describes novel pharmaceutical compositions that are able to overcome the drawbacks of the known art.
In U.S. Pat. No. 5,981,719 generally the polymers form the supporting structure of these microspheres, and the drug of interest is incorporated into the polymer structure.
U.S. Pat. No. 5,759,583: This invention provides a sustained release composition comprising a PLGA matrix, a bioactive agent, and a quaternary ammonium surfactant, in which the release profile of the bioactive agent from the PLGA matrix is controlled by the concentration of the quaternary ammonium surfactant.
U.S. Pat. No. 5,985,309: Particles incorporating a surfactant and/or a hydrophilic or hydrophobic complex of a positively or negatively charged therapeutic agent and a charged molecule of opposite charge for drug delivery to the pulmonary system, and methods for their synthesis and administration are provided.
U.S. Pat. No. 6,120,787 and WO 02/28370 A1: describe the use microparticles of a core material consisting of amylopectin-based starch and a biologically active substance that may be encapsulated with an outer release controlling shell.
In many of the examples from the prior art, there is a substantial initial burst effect, and very little protein released after that. Protein modifications, aggregation and loss of activity are also noticed in many formulations. Thus, given the current state of the art, there is a need for compositions and methods that effectively stabilize a variety of proteins to various physical and chemical environments, reduce the initial burst effects, and substantially release all the encapsulated protein. The present invention provides materials and methods to stabilize proteins, control the initial burst, and release the encapsulated protein in a controlled manner over a period of time.